Identification of an elusive spliceogenic MYBPC3 variant in an otherwise genotype-negative hypertrophic cardiomyopathy pedigree.

The finding of a genotype-negative hypertrophic cardiomyopathy (HCM) pedigree with several affected members indicating a familial origin of the disease has driven this study to discover causative gene variants. Genetic testing of the proband and subsequent family screening revealed the presence of a rare variant in the MYBPC3 gene, c.3331-26T>G in intron 30, with evidence supporting cosegregation with the disease in the family. An analysis of potential splice-altering activity using several splicing algorithms consistently yielded low scores. Minigene expression analysis at the mRNA and protein levels revealed that c.3331-26T>G is a spliceogenic variant with major splice-altering activity leading to undetectable levels of properly spliced transcripts or the corresponding protein. Minigene and patient mRNA analyses indicated that this variant induces complete and partial retention of intron 30, which was expected to lead to haploinsufficiency in carrier patients. As most spliceogenic MYBPC3 variants, c.3331-26T>G appears to be non-recurrent, since it was identified in only two additional unrelated probands in our large HCM cohort. In fact, the frequency analysis of 46 known splice-altering MYBPC3 intronic nucleotide substitutions in our HCM cohort revealed 9 recurrent and 16 non-recurrent variants present in a few probands (≤ 4), while 21 were not detected. The identification of non-recurrent elusive MYBPC3 spliceogenic variants that escape detection by in silico algorithms represents a challenge for genetic diagnosis of HCM and contributes to solving a fraction of genotype-negative HCM cases.

A cryptic splice-altering KCNQ1 variant in trans with R259L leading to Jervell and Lange-Nielsen syndrome.

Here we report an infant with clinical findings suggestive of Jervell and Lange-Nielsen syndrome (JLNS), including a prolonged QT interval (LQTS) and chronic bilateral sensorineural deafness. NGS analysis revealed one known heterozygous pathogenic missense variant, KCNQ1 p.R259L, previously associated with LQTS but insufficient to explain the cardioauditory disorder. In a screening of proximal intronic regions, we found a heterozygous variant, KCNQ1 c.1686-9 T > C, absent from controls and previously undescribed. Several splicing prediction tools returned low scores for this intronic variant. Driven by the proband's phenotype rather than the neutral predictions, we have characterized this rare intronic variant. Family analysis has shown that the proband inherited the missense and the intronic variants from his mother and father, respectively. A minigene splicing assay revealed that the intronic variant induced an additional transcript, arising from skipping of exon 14, which was translated into a truncated protein in transfected cells. The splice-out of exon 14 creates a frameshift in exon 15 and a stop codon in exon 16, which is the last exon of KCNQ1. This mis-spliced transcript is expected to escape nonsense-mediated decay and predicted to encode a truncated loss-of-function protein, KCNQ1 p.L563Kfs*73. The analysis of endogenous KCNQ1 expression in the blood of the proband's parents detected the aberrant transcript only in the patient's father. Taken together, these analyses confirmed the proband's diagnosis of JLNS1 and indicated that c.1686-9 T > C is a cryptic splice-altering variant, expanding the known genetic spectrum of biallelic KCNQ1 variant combinations leading to JLNS1.

A Pathogenic Galactosidase A Mutation Coexisting With an MYBPC3 Mutation in a Female Patient With Hypertrophic Cardiomyopathy.

The coexistence of GLA (Pro259Ser, c.775C>T) and MYBPC3 (c.1351+2T>C) mutations was found in a female patient with hypertrophic cardiomyopathy. Histology documented abundant vacuolisation with osmiophilic lamellar bodies and positive Gb3 immunohistochemistry. In the presence of a hypertrophic cardiomyopathy phenotype, the systematic search for unusual findings is mandatory to rule out a phenocopy.

A Novel Heterozygous Intronic Mutation in the FBN1 Gene Contributes to FBN1 RNA Missplicing Events in the Marfan Syndrome.

Marfan syndrome (MFS) is an autosomal dominantly inherited connective tissue disorder, mostly caused by mutations in the fibrillin-1 (FBN1) gene. We, by using targeted next-generation sequence analysis, identified a novel intronic FBN1 mutation (the c.2678-15C>A variant) in a MFS patient with aortic dilatation. The computational predictions showed that the heterozygous c.2678-15C>A intronic variant might influence the splicing process by differentially affecting canonical versus cryptic splice site utilization within intron 22 of the FBN1 gene. RT-PCR and Western blot analyses, using FBN1 minigenes transfected into HeLa and COS-7 cells, revealed that the c.2678-15C>A variant disrupts normal splicing of intron 22 leading to aberrant 13-nt intron 22 inclusion, frameshift, and premature termination codon. Collectively, the results strongly suggest that the c.2678-15C>A variant could lead to haploinsufficiency of the FBN1 functional protein and structural connective tissue fragility in MFS complicated by aorta dilation, a finding that further expands on the genetic basis of aortic pathology.

Phenotype and prognostic correlations of the converter region mutations affecting the ß myosin heavy chain.

OBJECTIVES: The prognostic value of genetic studies in cardiomyopathies is still controversial. Our objective was to evaluate the outcome of patients with cardiomyopathy with mutations in the converter domain of ß myosin heavy chain (MYH7). METHODS: Clinical characteristics and survival of 117 affected members with mutations in the converter domain of MYH7 were compared with 409 patients described in the literature with mutations in the same region. RESULTS: Twenty-five mutations were evaluated (9 in our families including 3 novel (Ile730Asn, Asp717Gly and Arg719Pro)). Clinical diagnoses were hypertrophic (n=407), dilated (n=15), non-compaction (n=4) and restrictive (n=5) cardiomyopathies, unspecified cardiomyopathy (n=11), sudden death (n=50) and 35 healthy carriers. One hundred eighty-four had events (cardiovascular death or transplant). Median event-free survival was 50±2 years in our patients and 53±3 years in the literature (p=0.27). There were significant differences in the outcome between mutation: Ile736Thr had fewer events than other mutations in the region (p=0.01), while Arg719Gln (p<0.01) had reduced event-free survival. CONCLUSIONS: Mutations in the converter region are generally associated with adverse prognosis although there are differences between mutations. The identification of a mutation in this particular region provides important prognostic information that should be considered in the clinical management of affected patients.

Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism.

INTRODUCTION: The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way. METHODS: Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[(3)H]glucose (2-[(3)H]DG). Statistical analysis was performed using the Mann-Whitney U test. RESULTS: NOC-12 liberates approximately ten times more NO2- than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[(3)H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[(3)H]DG than healthy cells. CONCLUSIONS: These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.

Screening mutations in myosin binding protein C3 gene in a cohort of patients with Hypertrophic Cardiomyopathy.

BACKGROUND: MyBPC3 mutations are amongst the most frequent causes of hypertrophic cardiomyopathy, however, its prevalence varies between populations. They have been associated with mild and late onset disease expression. Our objectives were to establish the prevalence of MyBPC3 mutations and determine their associated clinical characteristics in our patients. METHODS: Screening by Single Strand Conformation Polymorphisms (SSCP) and sequencing of the fragments with abnormal motility of the MyBPC3 gene in 130 unrelated consecutive HCM index cases. Genotype-Phenotype correlation studies were done in positive families. RESULTS: 16 mutations were found in 20 index cases (15%): 5 novel [D75N, V471E, Q327fs, IVS6+5G>A (homozygous), and IVS11-9G>A] and 11 previously described [A216T, R495W, R502Q (2 families), E542Q (3 families), T957S, R1022P (2 families), E1179K, K504del, K600fs, P955fs and IVS29+5G>A]. Maximum wall thickness and age at time of diagnosis were similar to patients with MYH7 mutations [25(7) vs. 27(8), p = 0.16], [46(16) vs. 44(19), p = 0.9]. CONCLUSIONS: Mutations in MyBPC3 are present in 15% of our hypertrophic cardiomyopathy families. Severe hypertrophy and early expression are compatible with the presence of MyBPC3 mutations. The genetic diagnosis not only allows avoiding clinical follow up of non carriers but it opens new possibilities that includes: to take preventive clinical decisions in mutation carriers than have not developed the disease yet, the establishment of genotype-phenotype relationship, and to establish a genetic diagnosis routine in patients with familial HCM.

Microalgae fiber optic biosensors for herbicide monitoring using sol-gel technology.

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol-gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol-gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 microg L(-1)) and the broadest dynamic calibration range (19-860 microg L(-1)) with IC(50) 125+/-14 microg L(-1). Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.

How eukaryotic algae can adapt to the Spain's Rio Tinto: a neo-Darwinian proposal for rapid adaptation to an extremely hostile ecosystem.

Microalgae contributed 60% of the total biomass in the extremely hostile (pH 2 and metal-rich waters) environment of Rio Tinto (which is used as a model for the astrobiology of Mars). These algae are closely related to nonextreme lineages, suggesting that adaptation to Rio Tinto water (RTW) must occur rapidly. Fitness from both the microalga Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa was inhibited when they were cultured in RTW. After further incubation for several weeks, D. chlorelloides survived, as a result of the growth of a variant that was resistant to RTW, but RTW-resistant cells did not appear in M. aeruginosa. A Luria-Delbrück fluctuation test revealed that D. chlorelloides RTW-resistant cells arose randomly by rare spontaneous mutations before the RTW exposure (1.38 x 10(-6) mutants per cell division). The mutants with a diminished fitness are maintained in nonextreme waters as the result of a balance between new RTW-resistant cells arising by mutation and RTW-resistant mutants eliminated by natural selection (equilibrium at c. 15 RTW-resistant per 10(7) wild-type cells). Rapid adaptation of eukaryotic algae to RTW could be the result of selection of RTW-resistant mutants occurring spontaneously in nonextreme populations that arrived fortuitously at the river in the past, or in the present continuously.

Xeno-implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects: an animal model.

Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno-transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankin's score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Mason's trichrome. Pig cells and pig genetic material were detected in the neo-synthesized tissue by immunohistochemical detection of SLA-II-DQ and polymerase chain reaction analysis of the gene SLA-II-DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo-synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline-like extracellular matrix. The neo-synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft-vs.-host rejections or infiltration by immune cells. In the neo-synthesized tissue, pig genetic material was detected in 27 +/- 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo-synthesized cartilage. We conclude that the xeno-transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.

Pig chondrocyte xenoimplants for human chondral defect repair: an in vitro model.

The objective of this study was to evaluate the use of cultured porcine chondrocyte xenotransplantation for the repair of human chondral defects. Two-millimeter-diameter defects were drilled into explants of femoral cartilage from healthy adult donors. No cells were implanted in the chondral defects of the control group, while pig chondrocytes from normal femoral cartilage were deposited into the treated chondral defects. Cartilage explants were cultured for 4, 8, and 12 weeks. Tissue sections were processed for standard histologic staining and immunostaining with monoclonal antibodies against types I and II collagen, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. The porcine origin of chondrocytes was confirmed using a specific pig monoclonal anti-CD46. Repair was only observed in the cell-treated defects. Mono- or bilayers of cells were detected after 4 culture weeks on the bottom of the defects, while after 8-12 weeks a repair tissue filled near 30-40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline-like region. This hypercellular region showed excellent bonding with the native cartilage, cells were located in numerous lacunae, and a high content of proteoglycans as indicated by an intense toluidine blue stain was observed. The repaired tissue showed positive immunostaining for both type I and II collagen, as well as chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. Positive staining for porcine anti-CD46 was localized exclusively in the neo-synthesized tissue. We conclude that xenotransplantation of pig chondrocytes can repair, in an in vitro model, defects in human articular cartilage.

Mitochondrial dysfunction in osteoarthritis.

In osteoarthritis (OA) a time or age dependent process leads to aberrant cartilage structure which is characterized by reduced number of chondrocytes, loss of existing cartilage extracellular matrix, the production of matrix with abnormal composition and pathologic matrix calcification. Because chondrocyte matrix synthesis and mineralization are modulated by the balance between ATP generation and consumption, the mechanism by which chondrocytes generate energy have been a topic of interest. The analysis of mitochondrial respiratory chain (MRC) activity in OA chondrocytes shows a significant decrease in complexes II and III compared to normal chondrocytes. On the other hand, mitochondrial mass is increased in OA, as demonstrated by a significant rise in CS activity. Furthermore, OA cells show a reduction in the mitochondrial membrane potential (deltapsim) as demonstrated by using the fluorescent probe JC-1. OA cartilage contains high number of apoptotic chondrocytes, and mitochondria play a key role in apoptosis. Interestingly, OA cartilages show markedly elevated Bcl-2 and caspasa-3 expression. This expression is also correlated with chondrocyte apoptosis and OA lesions. The pathogenesis of OA includes elaboration of increased amounts of NO as a consequence of up-regulation of chondrocyte-inducible NO synthase induced by IL-1, TNF-alpha and other factors. NO reduces chondrocyte survival and induces cell death with morphologic changes characteristic of chondrocyte apoptosis. NO reduces the activity of complex IV and decreases the deltapsim as measured as the ratio of red/green fluorescence. Furthermore, NO induces the mRNA expression of caspase-3 and -7, and it reduces the expression of mRNA bcl-2 and the bcl-2 protein synthesis. Some studies suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix. Mineral formation has been demonstrated in matrix vesicles (MV) and within mitochondria. Direct suppression of mitochondrial respiration promoted MV-mediated mineralization in chondrocytes. Regulation of MRC may be one of the signaling pathways by which NO modulates articular cartilage matrix biosynthesis and pathologic mineralization. After age 40, the incidence of OA in humans increases progressively with increasing age. Studies show a trend to statistic significance between the age and the reduction of complex I activity of human normal chondrocytes. However, the study of relation between age and deltapsim in normal chondrocytes do not demonstrate any significant correlation. It has been reported that as the number of population doublings increased, mitochondrial DNA was degraded and the number of mitochondria per chondrocyte decline. One approach for determining the role of mitochondria in OA is to determine the effects of the MRC inhibition and to compare them with the findings in OA. Inhibition of MRC with antimycin prevents the normal ability of TGFbeta to increase excretion of Pi, thereby worsening deposition of pathologic HA crystals. In chondrocytes, the inhibition of complex IV with NaN3 modified both the deltapsim and the survival of cells inducing apoptosis. Inhibition of complex I with rotenone increases the expression and synthesis of Bcl-2 and Cox-2, both effects are similar effects to produced by IL-1 in human chondrocytes.

Mitochondrial respiratory activity is altered in osteoarthritic human articular chondrocytes.

OBJECTIVE: Osteoarthritis (OA) is a degenerative rheumatic disease that is associated with extracellular matrix degradation and chondrocyte apoptosis in the articular cartilage. The role of mitochondria in degenerative diseases is widely recognized. We undertook this study to evaluate mitochondrial function in normal and OA chondrocytes and to examine age-related changes in mitochondria. METHODS: Mitochondrial function was evaluated by analyzing respiratory chain enzyme complexes and citrate synthase (CS) activities as well as changes in mitochondrial membrane potential (Delta Psi m). The activities of mitochondrial respiratory chain complexes (complex I: rotenone-sensitive NADH-coenzyme Q(1) reductase; complex II: succinate dehydrogenase; complex III: antimycin-sensitive ubiquinol cytochrome c reductase; and complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from OA and normal cartilage. Delta Psi m was measured by JC-1 using flow cytometry. Statistical analysis was performed using the Mann-Whitney U test and Student's t-test as well as several models of multiple linear regression. RESULTS: OA articular chondrocytes had reduced activities of complexes II and III compared with cells from normal cartilage. However, the mitochondrial mass was increased in OA. Cultures of OA chondrocytes contained a higher proportion of cells with de-energized mitochondria. We found no relationship between mitochondrial function and donor age either in normal or in OA chondrocytes. CONCLUSION: These findings suggest the involvement of mitochondrial function in the pathophysiology of OA. Cartilage degradation by OA and cartilage aging may be two different processes.